A method for delivering a substance into cells

ABSTRACT

A method for delivering a substance into cells, wherein the method comprises the following steps: a) providing the substance, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group, b) providing cells; and c) contacting the substance with the cells; wherein the method comprises the additional step of—adjusting a p H in an extracellular fluid to a p H of at least 7.7.

This invention was made with United States Government support underGrant No. R01-GM086801 awarded by the National Institutes of Health tothe Rensselaer Polytechnic Institute. The United States Government hascertain rights in this invention.

The invention relates to a method for delivering a substance into cells;a composition comprising a substance to be delivered to cells,particularly a skin treatment composition, more particularly in form ofa cream, a gel, an unguent, a lotion, a spray, a solution, in anemulsion, in liposomes or in microcapsules; a use of the composition fortreatment and/or prevention of a disease, diagnosis of diseases, as aresearch tool, as a targeting system, as a pharmaceutical composition oras a cosmetic composition; a use of the composition in the preparationof a medicament for treatment, prevention and/or diagnosis of a disease;a method for manufacturing the composition; and a kit comprising thecomposition.

Cell-penetrating peptides (CPP) consist of short amino acid sequences,usually rich in arginine amino acids, able to penetrate into almost anycell type, carrying with them cargoes such as proteins,oligonucleotides, and drugs. These short sequences are capable ofdirectly crossing the cell membrane in a seemingly energy-independentmanner. A fundamental question since the discovery of these peptides hasbeen how they do cross the plasma membrane to reach freely the interiorof cells. There is abundant evidence suggesting two main pathways:direct translocation across the plasma membrane and endocytosis. Bothpathways are described in general terms with fundamental questions leftunanswered. The most fundamental questions, at the heart of eitherpathway, are (a) how do these highly cationic peptides cross themembrane hydrophobic barrier and (b) what mechanism breaks the CPPs-,particularly RRPs-membrane strong ionic binding, once in the cytosol,detaching these peptides from the plasma or endosomal membrane.

Arginine-rich peptides (RRPs) are highly cationic and thereforehydrophilic. To directly cross the plasma membrane, or the endosomalmembrane, and diffuse freely in the cytosol these peptides must somehowovercome the strong barrier imposed by biological membranes. Thismembrane barrier has two distinctive components for RRPs, on one handthe peptides need to cross the hydrophobic barrier and on the other handthey need to detach from membranes overcoming their strong electrostaticbinding affinity for membrane components. Recently, a mechanism on howthese peptides could directly cross biological membranes and transportother molecules was proposed. This mechanism suggests that RRPs inducethe nucleation of small transient toroidal pores and then diffusethrough these pores towards the cytosolic side of the membrane. Atransient pore formation explains how these peptides are able todirectly cross into cells carrying along a wide range of cargoes.

However, a critical step for the nucleation of a pore in the known modelinvolves the membrane insertion of arginine amino acids to initiate thenucleation of a toroidal pore. The cost of insertion of thesearginine-rich peptides into model phospholipid bilayers seems too high(˜200 KJ/Mol). Therefore, this event might be too rare to be effectiveunless other factors help reduce this energetic cost. Furthermore, thismechanism does not explain how the peptides detach from the membraneafter the RRPs cross towards the cytosolic side of the membranes.

Early in vitro studies have suggested how RRPs could be absorbed into ahydrophobic environment. These studies have shown that arginine-richpeptides can be absorbed into a hydrophobic environment by binding tonegatively charged amphiphilic molecules forming a less polar or morehydrophobic complex. It has been shown that some of the molecules thatcan complex with guanidinium groups in this way are amphiphilic anionswith sulfur, phosphate or carboxyl groups, which are able to formbidentate hydrogen bonding with guanidinium groups. It has been shownthat these molecules can drive arginine-rich peptides into a hydrophobicenvironment such as chloroform and octanol. Using a typical, yetelegant, “U-tube” experiment with chloroform as a hydrophobic barrierbetween two buffer compartments, it has been shown that these complexescan mediate the transfer of reporter hydrophilic dyes across thehydrophobic phase from the cis buffer to the trans buffer. A similarflux of reporter dyes across vesicles containing amphiphilic anions wasalso observed. This suggests that these peptides might bind toamphiphilic anions forming complexes, which resemble inverted micelles,and that this complex can diffuse within hydrophobic environments.Consequently, the interior of these inverted micelles being hydrophilicmight capture and transport dyes across the hydrophobic barrier. Animportant aspect is that these experiments report the flux of dyesbetween two buffers separated by a hydrophobic barrier, not the flux ofthe RRPs themselves, which most of them are likely to just remaintrapped in the hydrophobic environment. Another important aspect of thisin vitro phenomenon is that it occurs spontaneously without therequirement of an external electrostatic potential.

In cells, on the other hand, it has been shown that the transmembranepotential is required for the cellular uptake of cell-penetratingpeptides. This membrane potential might be critical to reduce the plasmamembrane barrier. However, since the internal side of the bilayer shouldbe even more negative that the external side to maintain a negativepotential this should increase even further the affinity of the peptidestowards the bilayer. This opens the question of what mechanism allowsthe peptides to get released from the plasma membrane after crossing tothe cytosolic side.

Therefore, even if the peptides are somehow able to reach the cytosolicside of membranes there should be in place a mechanism to release thepeptides from the membranes. A distinctive aspect of these peptides istheir extremely high affinity for negatively charged membranecomponents. It has been shown that this ionic binding is so strong thatregular cell washes cannot remove the plasma membrane bound peptides.Removal of these peptides from the external side of the plasma membranerequires degrading them, a step usually done by trypsinization.Moreover, it has been suggested that not even degradation bytrypsinization is effective enough and that the addition of strongcounterions such as heparin to the wash solution is required tocompetitively bind to guanidinium groups and remove the membrane bounddegraded peptides. The transmembrane potential would in principle favoran even tighter peptide-membrane binding in the cytosolic side of theplasma membrane than in the exterior of the cell. Therefore, afundamental step in the uptake pathway is the mechanism under which thepeptides detach from the cell plasma membrane or the endosomal membraneto freely diffuse into the cytosol and reach the nucleus after reachingthe cytosolic side of these membranes.

With respect to the described limitations of delivering substances suchas peptides into cells, it is an object of the present invention toprovide a method for delivering efficiently various substances intocells.

These and other problems are solved by the subject matter of theattached independent claims.

The above objects of the invention are achieved by a method fordelivering a substance into cells according to claim 1, a compositioncomprising a substance to be delivered to the cells according to claim19, a use of the composition according to claim 25, a use of thecomposition according to claim 26, a method for manufacturing thecomposition according to claim 27, and a kit according to claim 28.

Preferred embodiments may be taken from the dependent claims.

The present inventors have surprisingly found how to use the pH gradientacross the plasma membrane of a target cell and thereby to increase theefficiency of delivery of a substance into target cells. Particularly,providing a high extracellular pH advantageously allows binding ofcompounds having at least one carboxyl functional group or carboxylatefunctional group coupled to a hydrophobic residue to extracellularcell-penetrating compounds (particularly CPPs), which comprise a basicamino functional group, (particularly at least one guanidinium group),and efficiently mediate the membrane transport of the cell-penetratingcompounds and their release into the lower-pH environment of thecytosol. In the context of the present invention it is important tounderstand that the basic amino functional group of the cell-penetratingcompound may particularly be part of a guanidinium functional group, forexample of RRPs. Thereby, the compounds with the carboxyl or carboxylatefunctional group may particularly be represented by the deprotonatedfatty acids of the target cell's membrane and/or, optionally, added as amediator compound, which allows further enhancement of the efficiency ofdelivery. The cell-penetrating compound thereby may particularlycomprise various cargoes such as small molecules, nucleic acids, peptidenucleic acids, proteins, oligonucleotides, inorganic particles, andliposomes, biomarkers, drugs and/or medically, pharmaceutically orcosmetically active substances.

According to the object of the invention, the present invention providesa method for delivering a substance into cells comprising the featuresof claim 1. Said method comprises at least the following steps:

-   -   a) providing the substance, wherein the substance comprises a        cell-penetrating compound comprising a basic amino functional        group,    -   b) providing cells; and    -   c) contacting the substance with the cells;        wherein the method comprises the additional step of    -   adjusting a pH in an extracellular fluid to a pH of at least        7.7, preferably of at least 7.8.

Thereby it is preferred that the extracellular fluid contains thesubstance, and particularly the cell-penetrating compound. Preferably, astep a) of providing the substance comprises providing the substance inan extracellular fluid.

Preferably, in step c) of contacting the substance with the cells, saidcontacting comprises delivering the substance into the cell. In otherwords, the method of the present invention preferably provides a pH inthe extracellular fluid, which is advantageously enhancing the deliveryof the substance into the cell, if the substance is contacted with thecells.

In some embodiments of the method according to the present invention,the method further comprises a step of applying the substance to asubject, wherein the method is a cosmetic or therapeutic method.

In some embodiments of the method, step b) of providing cells isconsisting of providing a suspension of cells, and in step c) contactingthe substance with the cells is consisting of creating acell-substance-suspension by combining the substance with the suspensionof cells, and wherein the pH in an extracellular fluid is anextracellular pH in the cell-substance-suspension. Preferably, theextracellular fluid is the cell-substance suspension.

In an embodiment the method may further comprise in step a) providing asuspension or solution of the substance in a pH buffer, particularly inan extracellular fluid being a pH buffer.

In connection with the present invention it is to be understood that thesubstance to be delivered may be advantageously delivered to nearly anytype of cell. The cell may be a eukaryotic or a prokaryotic cell. Thecell may be selected from the group comprising animal or human cells,mammalian cells, plant cells, bacterial cells or insect cells.Particularly a cell may also constitute a part of a multi-cellularorganism, in other words, a transgenic or non-transgenic organismcomprising at least one such cell. Particularly, some embodiments of thepresent invention comprise the use of the composition and/or methodsaccording to the present invention for treatment and/or prevention of adisease, diagnosis of diseases, and/or as a pharmaceutical compositionor as a cosmetic composition. In such embodiments the cell may also bepart of a multi-cellular organism represented by a subject subjected tosaid method or use, particularly a mammal, more particularly a human.

In some embodiments the method comprises creating acell-substance-suspension by combining the substance with the cells,particularly the suspension of cells.

According to the invention, the substance comprises a cell-penetratingcompound comprising a basic amino functional group, particularly atleast one guanidinium group and/or at least one amino group. Preferably,the cell-penetrating compound comprises at least one guanidinium groupand, optionally, at least one amino group. Preferably, thecell-penetrating compound is selected from peptides, proteins,carbohydrates, lipids and nucleic acids, oligonucleotides, peptidenucleic acids, inorganic particles, biomarkers, drugs, silicananoparticles, particularly silica nanoparticles having a cubicstructure, wherein amino groups are provided on the vertices of thecube. It is believed that at high extracellular pH the cell-penetratingcompound binds to plasma membrane molecules containing acidic groupssuch as e.g. fatty acids already present in the cell membrane therebyenhancing uptake significantly. It is to be understood that in mostmammalian cells the extracellular pH is about 7.4.

According to the invention, the extracellular pH is the pH in theextracellular fluid (ECF) outside of cells or in general terms the pHwhich is measurable outside of cells, such as e.g. the pH of thesubstance or composition, the pH outside of cells of the skin,particularly the pH of the skin of humans. The pH within cells is calledintracellular pH.

The substance can be or be provided as a homogeneous or heterogeneoussolution of various cell-penetrating compounds, particularlyguanidinium-rich compounds selected from peptides, proteins,carbohydrates, lipids and nucleic acids oligonucleotides, peptidenucleic acids, inorganic particles, biomarkers and drugs, silicananoparticles, particularly silica nanoparticles having a cubicstructure, wherein amino groups are provided on the vertices of thecube. It will be understood that the method can also be used to deliverany combination of the aforementioned compounds into cells. The solutionmay be a fluid, preferably a liquid or a gel such as an aqueous buffersolution. A pH buffer solution is an aqueous solution containing amixture of a weak acid and its conjugate base, or vice versa.

According to the invention, a higher pH is preferred. According to theinvention, the pH of the composition and/or the extracellular fluidand/or the suspension of cells is adjusted to a pH of at least 7.7,preferably of at least 7.8, still preferably of at least 8.0, stillpreferably of at least 8.5, more preferably of at least 8.75, mostpreferably of at least 9.0. Preferably, the pH is at maximum 9.5,preferably at maximum 10.0, more preferably at maximum 11.0, still morepreferably at maximum 12.0. Any suitable buffer may be used for thispurpose. Preferably, a pH buffer and/or a buffering agent may be addedto the suspension of cells to adjust the pH. A buffering agent canparticularly be either a weak acid or weak base or a chemicalformulation such as magnesium oxide or calcium carbonate that is addedto the cells, particularly the suspension of cells, to form a bufferedsolution with the desired pH. In some embodiments, the method and/orcomposition of the present invention is for treatment and/or preventionof a disease, diagnosis of diseases, as a pharmaceutical composition oras a cosmetic composition. Such methods may particularly in a step c) ofcontacting the substance with the cells, comprise a step of applicationof the composition and/or substance to a subject. Thereby it ispreferred that the substance and/or the composition and particularly abuffer is non-irritant and not toxic.

According to the invention, the cell-penetrating compound comprises abasic amino functional group. Amino functional groups contain a basicnitrogen atom with a lone electron pair. Preferably, the basic aminofunctional group of the cell-penetrating compound is part of aguanidinium functional group. It is to be understood that the presentinventors have found that a less number of —NH₂ groups can becompensated by an increased number of guanidinium functional groups.Accordingly, the term “basic amino functional group” as used herein,preferably comprises —NH₂ group or guanidinium functional group, ormixtures thereof. In a preferred embodiment, the cell-penetratingcompound is a peptide or a protein comprising a basic amino acidselected from arginine and lysine. Preferably, the number of basic aminofunctional groups is at least 1, still preferably at least 2, morepreferably at least 5, at least 6, at least 7, most preferably at least8 per 10 nm³ molecule of the cell-penetrating compound. Preferably, thenumber of basic amino functional groups is at least 1, still preferablyat least 2, more preferably at least 5, at least 6, at least 7, mostpreferably at least 8 per molecule of the cell-penetrating compound. Thecell-penetrating compounds of the substance that are ought to bedelivered into cells can also be described as biological oligomers.Particularly, the cell-penetrating compound can be a peptide,particularly a CPP, more particularly an RRP. The peptide may comprise aD-isomer or an L-isomer. Peptides which comprise polypeptides andoligopeptides are distinguished from proteins on the basis of size, andas an arbitrary benchmark can be understood to contain approximately 50or less amino acids, However, also other length of peptides areconsidered to be in the scope of the present invention. Preferably, acell-penetrating compound according to the present invention in the formof a peptide comprises 150 or less amino acid residues, 125 or lessamino acid residues, more preferably 105 or less amino acid residues.Preferably, a cell-penetrating compound according to the presentinvention in the form of a peptide comprises at least 85 amino acidresidues. In an embodiment the cell-penetrating compound is or comprisesa cyclic peptide. The use of a cyclic peptide may advantageously enhancethe cellular uptake. Proteins consist of one or more polypeptidesarranged in a biologically functional way. Either proteins or peptidescan be bound to ligands such as coenzymes and cofactors, or to anotherprotein or macromolecule (DNA, RNA, polysaccharides), or to complexmacromolecular assemblies.

Another preferred cell-penetrating compound comprises a DNA repairenzyme comprising a basic amino functional group. A particularlypreferred DNA repair enzyme is photolyase that is useful for repairingdamage caused by exposure of the cells to ultraviolet light. It isbelieved that photolyase is rich in guanidinium and amino groups.

According to another preferred embodiment, the cell-penetrating compoundcomprises a capping agent, and/or the method further comprises the stepof adding a capping agent, preferably to the cell-penetrating compound.Such capping agent may particularly comprise a guanidinium group whenusing a cell-penetrating compound comprising a peptide or a protein. Thecapping agent binds covalently or non-covalently to the N-terminus ofthe peptide or the protein. The additional guanidinium group provided bythe capping agent enhances the affinity of the peptide or the proteinfor the cell membrane and therefore facilitates cellular uptake. Apreferred capping agent is 4-guanidinobutyric acid.

The method may also employ cell-penetrating compounds such ascarbohydrates, lipids and nucleic acids. Nucleic acids are for exampleDNA or RNA, including messenger RNA, which DNA or RNA may be singlestranded or double stranded. In addition, a DNA-RNA hybrid whichcontains one strand of each may be utilized. Also a combination ofpeptides, proteins carbohydrates, lipids and/or nucleic acids can bedescribed as a substance to be delivered into cells.

According to a further aspect of the present invention, the methodcomprises the step of adding to the substance or cell-substance mix,particularly to the cell-substance-suspension- and/or to anextracellular fluid, preferably containing the substance, a mediatorcompound comprising at least one carboxyl functional group orcarboxylate functional group coupled to a hydrophobic residue.

The term “hydrophobic residue” as used herein, preferably means a carboncontaining backbone, preferably of at least 8 C-atoms. Preferably, acarbon containing backbone means a carbon-chain of at least 8 C-atoms.Thereby, said carbon containing backbone and/or carbon chain, may bepartially or completely saturated or unsaturated.

It is believed that at high extracellular pH the mediator compoundcomprising a carboxylate functional group binds the cell-penetratingcompound comprising a basic amino functional group, mediates theirmembrane-transport across the hydrophobic core of the cell membrane andreleases into the lower pH environment of the cytosol. This results in avery efficient way of delivering the substance into virtually any cell.It is immediately understood by a person skilled in the art that alsothe cell to which the substance is to be delivered may comprisecompounds comprising at least one carboxyl functional group orcarboxylate functional group coupled to a hydrophobic residue, forexample, in the form of fatty acids of the cell membrane. However, theaddition of a mediator compound can advantageously increase theefficiency of the delivery.

The mediator compound comprising a carboxyl/carboxylate functional groupattracts and binds arginine and/or lysine rich cell-penetrating peptidesor proteins and carries these peptides or proteins across the cellmembrane into the cytosol.

It is assumed that the cell-penetrating compound interacts with the cellmembrane, thereby reducing the energetic cost of insertion of thesubstance into the plasma membrane. The mediator compound comprising atleast one carboxyl functional group or carboxylate functional groupcoupled to a hydrophobic residue which is added to thecell-substance-suspension is preferably an anionic amphiphilic moleculethat electrostatically binds to the substance to be delivered into thecells.

Typical mediator compounds display high solubility in 1-octanol and lowsolubility in water. Preferably, the distribution of a mediator compoundin a mixture of water and 1-octanol is more than 50 mol-% in the1-octanol phase, most preferred more than 80 mol-% in the 1-octanolphase. The hydrophobicity of the mediator compound may be enhanced bycoupling a functional group present in the mediator compound, such as anamine group, to a protective group such as Fmoc, e.g. 12 Ado-OH:

Preferred mediator compounds are compounds that are naturally occurringin cell membranes, such as fatty acids. Fatty acids increase thefluidity of membranes and can be both negatively charged and neutralwithin a physiological pH range. Particularly preferred mediatorcompounds are saturated and non-saturated fatty acids, saturated andnon-saturated hydroxy fatty acids, dicarboxy acids, aromatic acids andsalts thereof. A mediator compound preferably has a pKa of 2 or more, 3or more, 4 or more, 5 or more, more preferably of 7 or more, mostpreferably of 8 or more. The pKa is preferably at most 12, morepreferred at most 11, most preferred at most 10.

Particularly, the molar ratio of mediator compound per cell-penetratingcompound depends on the desired mediator compound and the particularcell-penetrating compound. In a preferred embodiment the molar ratio ofmediator compound to cell-penetrating compound is at least 1.0, i.e. onemediator per cell penetrating compound. In other words, the ratio ofmediator compound to cell-penetrating compound preferably is 1 or more,still preferably 1.5 or more, 2.0 or more, 2.5 or more, more preferably3.0 or more, 4.0 or more, most preferably 5.0 or more.

In some embodiments of the method and the composition of the presentinvention various cargoes such as small molecules, nucleic acids,peptide nucleic acids, proteins, oligonucleotides, inorganic particles,and liposomes are to be delivered to the cell to mediate a desiredeffect, e.g. a therapeutic, medical or cosmetic effect, as a biomarker,drug and/or a medically, pharmaceutically or cosmetically activesubstance. Such cargoes as used herein may particularly be comprised inthe cell-penetrating compound and/or added as a, or a part of a mediatorcompound.

Most preferred mediator compounds, which particularly may be usefulcargoes, are compounds having a desired effect, e.g. a therapeutic,medical or cosmetic effect, in the cell, such as artemisinin andderivatives, such as artesunate, folic acid, levomefolic acid,levothyroxine, curcumin,(1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione,atorvastatin,(3R,5R)-7-[2-(4-Fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoicacid, S-Adenosyl methionine,(2S)-2-Amino-4-[[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl-methylsulfonio]butanoate,salicylic acid, 2-Hydroxybenzoic acid, lipoic acid,(R)-5-(1,2-Dithiolan-3-yl)pentanoic acid,5,10-methylenetetrahydrofolate, D-α-tocopherol succinate, a tocopherylacid succinate, ibuprofen, dexibuprofen, and salts thereof. Furtherpreferred mediator compounds are peptidomimetics comprising ahydrophobic backbone and a carboxyl functional group,biphenyl-4-carboxylic acid, benzoic acid, ricinoleic acid,(2E)-18-hydroxyoctadec-2-enoic acid 18-hydroxyoctadec-9-enoic acid,6-hydroxyhex-3-enoic acid, trans-3-hydroxyhex-4-enoic acid,(E)-10-hydroxydec-2-enoic acid, (2E)-9-hydroxydec-2-enoic acid,8-hydroxyoctanoic acid, 9-hydroxynonanoic acid, 11-hydroxyundecanoicacid, 12-hydroxydodecanoic acid, 15-hydroxypentadecanoic acid,16-hydroxyhexadecanoic acid, 17-hydroxyheptadecanoic acid,2-hydroxyheptadecanoate,(5Z,8Z,11Z,14Z)-19-hydroxynonadeca-5,8,11,14-tetraenoic acid,(10E,12E)-14-hydroxy-10,12-nonadecadienoic acid,20-hydroxyeicosatetraenoic acid,(5Z,8Z,10E,14Z)-12-hydroxyicosa-5,8,10,14-tetraenoic acid,16-Hydroxyhenicosa-2,4,6-trienoic acid,18-bromo-16-hydroxytricosa-8,17,19-trien-4,6-diynoic acid, 12-Ado-OH,ascorbic acid, azelaic acid, sebacic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid,(6aR,11aS,11bR)-10-Acetyl-11-hydroxy-7,7-dimethyl-2,6,6a,7,11a,11b-hexahydro-9H-pyrrolo[1′,2′:2,3]isoindolo[4,5,6-cd]indol-9-oneand salts thereof.

Generally, the interior of cells is maintained at a lower pH than theexterior. This pH gradient across the cell plasma membrane can probablyincrease the deprotonation of above-mentioned acids in the extracellularlayer of the plasma membrane at higher pH. Presumably, the mediatorcompound neutralizes the charge of the substance and reduces theenergetic cost of the insertion of the substance into the hydrophobiccore of the lipid bilayer of the cell plasma membrane. This insertionleads to the formation of a channel across the cell plasma membranefacilitating the transport of the substance across the cell plasmamembrane. It was surprisingly found that the binding affinity betweenthe mediator compound and the substance to be delivered into cells isreduced when the substance has reached the cytosolic side of themembrane. It is believed that in contact with the lower cytosolic pH,the mediator compound becomes protonated and neutrally charged, thesubstance is released from the plasma membrane into the cytosol and thechannel closes. Experiments suggest that also the mediator compound isreleased into the cytosol to a significant extent, thus enabling thetransport of therapeutically effective mediator compounds into thecytosol.

In a particularly preferred embodiment, the cells are pre-incubated withthe mediator compound before the cell-substance-suspension is created.It was surprisingly observed that cells enriched with the mediatorcompound displayed enhanced substance uptake efficiency. It is assumedthat this effect is due to enrichment of the plasma membrane with themediator compound. Alternatively or additionally, the substance can bepre-incubated with the mediator compound before thecell-substance-suspension is created.

The substance that is to be delivered into cells, particularly thecell-penetrating compound, can also be a cell-penetrating peptide,particularly an RRP. Cell-penetrating peptides (CPPs) are short peptidesthat facilitate cellular uptake of various additional agents,particularly cargo molecules, such as nanosize particles, small chemicalmolecules and fragments of DNA. The additional agent is associated withthe peptides either through chemical linkage via covalent bonds orthrough non-covalent interactions. Cell-penetrating peptides typicallyhave an amino acid composition that either contains a high relativeabundance of positively charged amino acids such as lysine or arginineor has sequences that contain an alternating pattern of polar/chargedamino acids and non-polar, hydrophobic amino acids. These two types ofstructures are referred to as polycationic or amphipathic, respectively.A third class of cell-penetrating peptides are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups.

In a preferred embodiment, in order to use the substance fortransporting an additional agent, for example DNA, RNA or a peptide intocells, the cell-penetrating compound comprises a linker moiety. Thelinker moiety is preferably a peptide or a nucleotide linker that isable to conjugate the additional molecule covalently or non-covalentlyconjugated to the cell-penetrating compound. Typical additional agentsare selected from small molecules, nucleic acids, peptide nucleic acids,peptides, proteins, nucleotides, oligonucleotides, inorganic particlesand liposomes. Preferably, the additional agent comprises at least onecarboxyl functional group or carboxylate functional group. In case ofpeptides or proteins, the additional agent comprises at least one acidicamino acid selected from aspartate/aspartic acid and glutamate/glutamicacid. Particularly, the additional agent can be selected from the groupcomprising biomarker, drug, medically, pharmaceutically and cosmeticallyactive substances.

In another aspect, the invention relates to a composition comprising asubstance to be delivered to the cells, according to claim 19. Inconnection therewith it is to be understood that any feature describedherein with regard to the method of the method according to the presentinvention may also be a feature of the composition of the presentinvention and vice versa.

In a composition according to the present invention the substancecomprises a cell-penetrating compound comprising a basic aminofunctional group, and wherein the pH of the composition is at least 7.7,preferably at least 7.8. In an embodiment the cell-penetrating compoundis selected from the group comprising peptides, proteins, carbohydrates,lipids and nucleic acids. In some embodiments the composition comprisesa suspension of the substance in pH-buffer, particularly in anextracellular fluid being a pH buffer. The composition may also comprisea mediator compound comprising at least one carboxyl functional group orcarboxylate functional group coupled to a hydrophobic residue.Particularly, the composition can be provided in the form of a cream, agel, an unguent, a lotion, a spray, an aerosol, a solution, in anemulsion, in liposomes or in microcapsules. Particularly the compositionprovided for skin treatment. Particularly, the present inventionprovides a skin treatment composition in a form of a cream, a gel, anunguent, a lotion, a spray, an aerosol, a solution, in an emulsion, inliposomes or in microcapsules, wherein the composition comprises amediator compound comprising at least one carboxyl functional group orcarboxylate functional group coupled to a hydrophobic residue, asubstance to be delivered to the cells of the skin and a pH buffer,wherein the pH of the composition is at least 7.7, preferably at least7.8. The skin treatment composition allows for uptake of substances andpreferably additional active agents bound to the substance into the skincells thereby providing a desired effect.

The composition of the present invention may be used for the treatmentand/or prevention of a disease, diagnosis of diseases, as a researchtool, as a targeting system, as a pharmaceutical or cosmeticcomposition. Preferably, the substance to be delivered to the cellscomprises a pharmaceutically active compound. However, the substance canalso be used as a carrier for delivering an additional pharmaceuticalactive agent, such as DNA, a protein, a peptide or another biomoleculeto the cells. In this case, the additional active agent is covalently ornon-covalently linked to the substance as described above. Apharmaceutical active agent is a compound contained in a pharmaceuticaldrug that is biologically active.

Furthermore, it is preferred that the composition comprises apharmaceutical acceptable carrier, filler, bulking agent, disintegrant,stabilizer, binder, humectant, extender, emulsifying agent, dissolutionretarder, absorption enhancer, preservative, antioxidant, wetting agent,adsorbent, lubricant or a combination thereof. The term“pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a mammal in need of the active compound.The term “pharmaceutically acceptable carrier” as used herein means anymaterial or substance present in a formulation in order to facilitateits application or dissemination to the locus to be treated, forinstance by dissolving, dispersing or diffusing said composition, and/orto facilitate its storage, transport or handling without impairing itseffectiveness.

As a result, the composition can be adjusted to the specific requirementof multiple biologically different diseases. Particularly, theconcentration or amount of cell-penetrating compound can be adjusted tothe desired needs and in order to achieve the desired effect. Forexample, the concentration or amount of cell-penetrating compound can beadjusted to allow for efficient transport into skin cells.

In some embodiments the composition of the present invention is for theuse in the application to a subject, e.g. for the treatment and/orprevention of a disease, diagnosis of diseases, as a pharmaceutical orcosmetic composition. In such embodiments, the composition may beadministered to the subject by applying the composition of the presentinvention to the subject. Particularly, with regard to the applicationto the skin, the composition may be applied to the skin and thus to theskin cells. However, other forms of application are also consideredherein, for example, the composition may be locally injected at a tumorsite. Accordingly, a method of the present invention may comprise a stepof applying the composition to a subject. Particularly, said applicationstep may be part of step c) of contacting the substance with thecells—depending on the desired application. For example, a cosmetictreatment of the skin may comprise application of the composition to theskin, in the form of a cream, a gel, an unguent, a lotion, a spray, anaerosol, a solution, in an emulsion, in liposomes or in microcapsules,and thereby contacting the composition, particularly the substance, withthe skin cells.

The active compound can thereby be delivered specifically to the desiredlocations, for example the skin of a subject. Examples of types ofactive compounds that may be transported to the target cells,particularly skin cells, by the composition comprise analgesics,anesthetics, antianginals, antifungals, antibiotics, anticancer drugs,anti-inflammatories, anthelmintics, antidepressants, antidotes,antiemetics, antihistamines, antihypertensives, antimalarials,antimicrotubule agents, antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, antisignaling agents,antiarthritics, antithrombin agents, antituberculotics, antitussives,antivirals, appetite suppressants, cardioactive drugs, chemicaldependency, drugs, cathartics, chemotherapeutic agents, coronary,cerebral or peripheral vasodilators, contraceptive agents, depressants,diuretics, expectorants, growth factors, hormonal agents, hypnotics,immunosuppression agents, narcotic antagonists, parasympathomimetics,sedatives, stimulants, sympathomimetics, toxins and tranquilizers.

In another aspect, the present invention is related to a use of thecomposition according to the present invention in the preparation of amedicament for treatment, prevention and/or diagnosis of a disease.

In another aspect, the present invention is related to a method formanufacturing

-   -   the composition according to the present invention. Said method        for manufacturing comprises at least the following steps:    -   providing a substance comprising a cell-penetrating compound        comprising a basic amino functional group, and    -   adjusting the pH of the composition to at least 7.7, preferably        at least 7.8.

In another aspect, the present invention is related to a kit comprisingin (a) suitable container(s) at least a composition according to thepresent invention, and optionally a package insert. Preferably, thecomposition is contained in a ready-to-use form.

The invention is illustrated but not limited by the following examples.

EXAMPLES Example 1: Uptake of TAMRA Labeled HIV 1 TAT-Peptide in HeLaLiving Cells

HeLa cells where seeded at 60% confluence in a tissue culture treated 6channel μ-Slide VI (from Ibidi GmbH, Germany) 24 hr before peptidetreatment. The uptake imaging at different pHs was done by washing twotimes with HEPES buffers (140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mMglycine, pH adjusted with NaOH or HCl) at the pH of interest. Thesubstance, here the HIV 1 TAT-peptide was provided in HEPES buffer. Thebuffer solution was replaced with said suspension, comprising the HEPESbuffer with the peptide added at a final concentration of 2 μM, andadjusted to the desired pH value. The sample was taken to the microscopeand imaged at equally spaced intervals of 2 min. This was donesimultaneously for pH 6, 7.5 and 9 to compare the relative peptideuptake side by side. HeLa cells were imaged by swapping at each timepoint between two objectives, an ×60 and an ×20 immersion oil. This wasdone to be able to perform two types of time-lapse analysis over thesame sample to compute the cellular uptake of the TAT peptide. With the×20 objective is possible to simultaneously visualize several cells butis not easy to separate the fluorescence intensity from internalizedpeptide from the fluorescent intensity of membrane bound peptide. Withthe ×60 objective a fewer number of cells are visualized but therelative intensity of free peptide can be computed by measuring thefluorescence intensity of peptides accumulated at the nucleolus, sincepeptide bound to the cell plasma membrane or trapped in endosomes cannotreach the nucleolus. Using the ×20 images, the uptake was computed bymeasuring the average background fluorescence intensity in an area ofthe images without cells and subtracting this value from the averageintensity of the whole image. Using the ×60 images, the uptake wascomputed by measuring the average background fluorescence intensity inan area without cells and subtracting this value from the averageintensity in the nucleus. The nucleus area was obtained using the DICchannel. This experiment was repeated 3 times and the average and thestandard error plotted. In each experiment, after 30 min the cells werewashed with DMEM cell culture media and calcein was added to detect cellviability at a final concentration of 5 μM. In live cells, thenon-fluorescent calcein AM is converted to green-fluorescent calcein,after acetoxymethyl ester hydrolysis by intracellular esterases. Thiswas incubated for 30 min and then imaged. Cell viability was alsoassessed using the DIC images used to detect the cell morphology alongthe experiments. To further evaluate the viability of cells after uptakeof the TAT at pH 9, cell division was monitored for 16 hours.

The results are shown in FIG. 1. FIG. 1 shows the fluorescence intensityof TAT (2 μM) uptake in living cells at pH 6, 7.5 and 9 vs. time.Accordingly, at the concentration of 2 μM of the TAT peptide there is nouptake at pH 6 and 7.5, most cells kept at pH 9 display a significantuptake within this time interval (30 min).

Example 2: Comparison of Different Mediator Compounds on the UptakeAmino-Rich and Guanidinium-Rich Cell-Penetrating Compounds in LivingCells

A comparative test of cellular penetration of different cell-penetratingcompounds was performed in the presence and absence of differentmediator compounds at low and high pH. The cell-penetrating compounds (2μM) and the mediator compounds (6 μM) were mixed in Hepes buffers at lowpH (6.5) and high pH (8.5).

The cell media (DMEM) was removed and exchanged by these solutions andincubated for 30 min. Then the solutions were removed and the cells wereembedded back in cell culture media (DMEM) and imaged. The efficiency ofuptake in the different conditions was determined.

The results are shown in Table 1 below. The determined efficacy isdepicted with increasing efficacy from “−” no uptake detectable, “+” lowefficiency of uptake detectable, “++” moderate efficiency of uptakedetectable, “+++” strong de efficiency of uptake detectable, “++++” verystrong efficiency of uptake detectable.

TABLE 1 Efficiency of uptake in different conditions. Mediator 1:Mediator 2: no mediator Curcumin Levothyroxine low pH high pH low pHhigh pH low pH high pH of 6.5 of 8.5 of 6.5 of 8.5 of 6.5 of 8.5 Peptide1: TAT peptide − + − ++ − ++ labeled with TAMRA Peptide 2: R10 − + − +++− +++ labeled with FITC cyclic Peptide: cR10 − ++ − ++++ − ++++ labeledwith FITC DNA repair enzyme − − − ++ − ++ Nanoparticle with amino + ++ ++++ + +++ functional groups: Cube octameric silsesquioxanes (COSS)labeled with FITC. Comparative example peptide − − − − − − without aminofunctional groups: TAMRA-Ahx- LGQQQPFPPQQPY

Example 3: Cellular Uptake in Fatty Acid Enriched Cells

A HEPES buffer at pH 7.5 was mixed with 0.2% volume of oleic acid. Cellswere washed twice and incubated for 15 min with this buffer. Next, cellswere washed once with a HEPES buffer at pH 7.5 (without oleic acid) andthe TAT peptide mixed with this last buffer was added at differentconcentrations. Cells were washed after 5 min with DMEM cell culturemedia two times, media plus calcein was added to monitor for enzymaticactivity and the cells were imaged.

The results are shown in FIG. 5. It may immediately taken therefrom thatfatty acid enriched cells display a much higher uptake efficiency thanthe control cells and most cells are viable as indicated by theirmorphology and their enzymatic activity. Therefore, enriching the plasmamembrane with fatty acids enhances the binding and uptake ofarginine-rich peptides.

Example 4: Peptides Absorption into a Hydrophobic Phase as a Function ofpH

TAMRA labeled TAT peptides were diluted in HEPES buffer (140 mM NaCl,2.5 mM KCl, 5 mM HEPES, 5 mM glycine, pH adjusted with NaOH or HCl) to afinal concentration of 10 μM at each pH. 150 μl of the buffer-peptidemix was added to 146 μl of octanol plus 4 μl of a compound selected fromoctanol, oleic acid, acetic acid, mono-N-dodecylphosphate, sodiumdodecylsulfate, lithocholic acid, sunflower oil, castor oil and oliveoil. Each pH mix was vortexed for 5 min and centrifuged for 2 min with acentripetal force of 2200 g to separate the octanol from aqueous phases.The octanol phase in contact with each pH buffer was then extracted witha pipette and mixed with a buffer at pH 4, which was also vortexed andcentrifuged. In this way the fraction of peptide previously absorbed inthe octanol phase was reabsorbed in the pH 4 buffer and in this case theoctanol phase (with little or no traces of the TAT peptides) was againremoved with a pipette and discarded. The peptide in each buffersolution was measured using a fluorescent spectrometer, using as areference a buffer solution of 10 μM peptide at pH 4, measuring therelative fluorescent intensity emission between the reference andsolution and the solution of interest, exciting with a laser wavelengthof 543 nm and measuring the emission wavelength of 575 nm. Similarly thepeptides that remained in the aqueous phase were compared to a referencesolution of 10 μM peptide at each pH without having been in contact withthe octanol phase.

The results are shown in FIG. 2. In the left column of FIG. 2 are shownsnapshots of microcentrifuge tubes containing the different hydrophobicphases in contact with the aqueous buffers at different pHs. FIG. 2ashows that in the absence of oleic acid as well and in the presence ofacetic acid TAT does not enter the hydrophobic phase. FIG. 2b shows thatphosphate and sulfur groups containing compounds remain bounded to theTAT peptide and partition into the hydrophobic phase at every pH.Lithocholic acid displays a similar behavior as oleic acid although thedeprotonation in this case is shifted to a higher pH. FIG. 2c showspartition of the TAT peptide into three distinct types of naturalvegetable oils: sunflower oil, castor oil and olive oil. Sunflower oildisplays a behavior consistent with a composition of only triglyceridesdisplaying no absorption of the TAT peptide in the hydrophobic phase.Castor oil behaves as having also free fatty acids showing an absorptionbehavior similar to oleic acid. Olive oil displays absorption of the TATpeptide at the interface for every pH revealing the presence ofphospholipids.

The results shown in FIG. 2c open a new approach for detecting ifnatural oil has expired or if it has been adulterated by mixing it witha different oil.

It is well known that oxidized or aged olive oil can be detected bydetermining the content and nature of free fatty acids. Therefore, theabsorption of a peptide at high pHs into a hydrophobic phase wouldchange.

An olive oil that is adulterated with tea tree oil can be detected bytwo significant changes depending on the percentage of tea tree oiladded. Firstly, the absorption of peptides at the interface between theoil and the aqueous buffer changes, since the concentration ofphospholipids naturally present in olive oil changes, and secondly, theamount of peptide absorbed at high pH also changes, since the mixreduces the amount of free fatty acids.

Using a proper calibration, the above methods offer a simple, immediateand cheap way to detect oil that has been adulterated or that isexpired. Particularly, the present application in a further aspect isrelated to a method for determining the state, particularly the quality,of an oil. Such method particularly comprises at least the followingsteps: providing a sample of the oil, applying an aqueous buffer to theoil sample, adding a peptide according to the present invention to thesample, and determining the amount of peptide absorbed at high pH and/ordetermining the absorption of peptides at the interface between the oiland the aqueous buffer. The absorption of the peptides in olive oil andcanola oil or other oils is very different. Therefore, knowing how thepeptide is absorbed at different pHs allows a person skilled in the artto conclude on the quality of the oil, and even if the oil has beenadulterated.

As apparent from FIG. 2, the peptides can be easily used to detectdifferent kind of oils and compositions within the oils.

Example 5: Efficacy of the Composition of the Present Invention as aSkin Treatment

For determining the efficacy of the composition of the presentinvention, a composition was manufactured comprising a buffer solutionat a pH higher than 7.8 an RRP at a concentration of 10 micro Molarslabeled with a fluorescent die.

The composition is tested in a clinical study, wherein volunteers donate30 mm² of skin extracted by a punch biopsy. The skin sample is placed ina tube with the stratum corneum exposed to air while the other kinlayers are submerged into cell culture media. A drop of the buffersolution containing the cell-penetrating compound is applied on top ofthe stratum corneum for 1 hr. Then the area is washed 3 times with PBS,the skin is sectioned with a scalp and imaged at a confocal microscope.It can be seen that the cell-penetrating substance is able to cross thestratum corneum and get inserted in cells within several layers belowthe stratum corneum.

The efficacy can be clearly seen as the fluorescently labeledguanidinium-rich peptide is able to cross the stratum corneoum and reachthe interior of cells. In the cells the nucleolus is also labeledindicating the peptide is not entrapped in endosomes and is consequentlyimmediately bioavailable.

Example 6: Uptake of Compounds at Different pHs

FIG. 3 shows the uptake of other compounds at different pHs, showingthat the pH enhances penetration of guanidinium and amino groups intothe cells.

It can be immediately taken therefrom that increasing the extracellularpH consistently increases the transduction efficiency of arginine richpeptides with different structures, lengths and chirality. Fluorescenceimages show the uptake of the peptides listed in Table 1 (2 μM) inliving cells (HeLa) at pH 6, 7.5 and 9 after 30 min. For each pH isshown the fluorescent confocal image of the peptides (green) and animage composed of an overlay of the DIC, the peptides and the nucleus(red). Cells permanently expressing Proliferating Cell Nuclear Antigen(PCNA) labeled with Cherry or GFP were used to facilitate the detectionand visualization of confocal planes across the nucleus. This helped toeasily classify and count cells containing transduced peptides from thecells that only contain membrane bound peptides. Although, this is clearwhen the peptides are in D form (R10 or cR10) since they are stable anddistinctly label the nucleolus, the peptides in L form are beingactively degraded and the signal quickly redistributes morehomogeneously within the cell making more challenging to distinguishmembrane bound peptides form internalized peptides. Cells were countedas positive when the peptide signal co-localized with the PCNA signal.The percentage of cells counted with intracellular peptide at pH 9 wasconsistently larger relative to pH 6 and 7.5 for all arginine richpeptides, while the poly-lysine peptide K10 displays no uptake at allpHs. The uptake efficiency also increases with the number of arginineamino acids and by cyclization. The images where acquired with a ×60objective magnification. Each experiment was repeated 3 times and thepercentage represents the average over more than 400 cells in each case.Scale bar 15 μm.

Example 7: Delivery of an Arginine and Amino Rich Peptide

FIG. 4 shows the delivery of an arginine and amino rich peptide, herethe TAT peptide, transporting a florescent molecule across the skin.

A skin sample was obtained from a healthy individual and a drop of thecompound in a buffer at pH 9 mixed with ricinoliec acid (the compoundand the fatty acid at a concentration of 20 μM) was immediately appliedon the skin surface for 60 min. The dermis was kept permanently incontact with cell media supplemented with nutrients to keep the tissuealive. The living sample was then washed, fractioned with a scalp andimaged with a confocal microscope specially designed to image livingtissue.

It can be immediately taken from FIG. 4 that the fluorescently labeledmolecule is transported across the stratum corneum and reaches theepidermis and dermis layers and the cells interior (distributed in thecytosol and the cell nucleus). The different layers are schematicallyshown as a reference. The distribution gradient of the drug accumulationis immediately evident, as it goes deeper into the skin layers,naturally with higher concentration in the upper layers.

One with ordinary skill in the art will recognize from the provideddescription and examples that modifications and changes can be made tothe various embodiments of the invention without departing from thescope of the invention defined by the claims and their equivalents.

The features of the present invention disclosed in the specification,the claims, examples and/or the figures may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

What is claimed is:
 1. A method for delivering a substance into cells,wherein the method comprises the following steps: a) providing thesubstance, wherein the substance comprises a cell-penetrating compoundcomprising a basic amino functional group, b) providing cells; and c)contacting the substance with the cells; wherein the method comprisesthe additional step of adjusting a pH in an extracellular fluid to a pHof at least 7.7.
 2. The method according to claim 1, wherein thecell-penetrating compound is selected from the group comprisingpeptides, proteins, carbohydrates, lipids and nucleic acids.
 3. Themethod according to claim 1, wherein the cells are chosen from the groupcomprising mammalian cells, plant cells, bacterial cells or insectcells.
 4. The method according to claim 1, wherein in step b) providingcells is consisting of providing a suspension of cells, and wherein instep c) contacting the substance with the cells is consisting ofcreating a cell-substance-suspension by combining the substance with thesuspension of cells, and wherein the pH in an extracellular fluid is anextracellular pH in the cell-substance-suspension.
 5. The methodaccording to claim 1, further comprising in step a) providing asuspension or solution of the substance in a pH buffer, particularly inan extracellular fluid being a pH buffer.
 6. The method according toclaim 1, wherein the method further comprises adding to the substanceand/or to an extracellular fluid and/or the cell-substance-suspension, amediator compound comprising at least one carboxyl functional group orcarboxylate functional group coupled to a hydrophobic residue.
 7. Themethod according to claim 1, wherein the basic amino functional group ofthe cell-penetrating compound are part of a guanidinium functionalgroup.
 8. The method according to claim 1, wherein the cell-penetratingcompound is a peptide or a protein comprising a basic amino acidselected from arginine and lysine.
 9. The method according to claim 1,wherein the cell-penetrating compound is a peptide or a protein and themethod further comprises the step of adding a capping agent comprising aguanidinium group.
 10. The method according to claim 1, wherein thecell-penetrating compound comprises a DNA repair enzyme comprising abasic amino functional group.
 11. The method according to claim 1,wherein the cell-penetrating compound comprises a linker moiety.
 12. Themethod according to claim 11, wherein the linker moiety is a peptidelinker or a nucleotide linker.
 13. The method according to claim 1,wherein an additional agent is covalently or non-covalently conjugatedto the cell-penetrating compound, the additional agent being selectedfrom small molecules, nucleic acids, peptide nucleic acids, peptides,proteins, nucleotides, oligonucleotides, inorganic particles andliposomes.
 14. The method according to claim 13, wherein the additionalagent is selected from the group comprising biomarker, drug, medically,pharmaceutically and cosmetically active substance.
 15. The methodaccording to claim 13, wherein the additional agent comprises at leastone carboxyl functional group or carboxylate functional group.
 16. Themethod according to claim 15, wherein the additional agent is a peptideor a protein comprising an acidic amino acid selected fromaspartate/aspartic acid and glutamate/glutamic acid.
 17. The methodaccording to claim 1, wherein the mediator compound is selected from thegroup consisting of saturated and non-saturated fatty acids, saturatedand non-saturated hydroxy fatty acids, dicarboxy acids, aromatic acids,and salts thereof.
 18. The method according to claim 1, wherein thecells and/or the substance are pre-incubated with the mediator compoundbefore the cell-substance-suspension is created and/or before in step c)the substance is contacted with the cells.
 19. The method according toclaim 1, wherein step c) of contacting the substance with the cells,comprises delivering the substance into the cell.
 20. The compositionaccording to claim 19, wherein the cell-penetrating compound is selectedfrom the group comprising peptides, proteins, carbohydrates, lipids andnucleic acids.
 21. The composition according to claim 19, wherein thecomposition comprises a suspension of the substance in pH-buffer,particularly in an extracellular fluid being a pH buffer.
 22. Thecomposition according to claim 19, wherein the composition comprises amediator compound comprising at least one carboxyl functional group orcarboxylate functional group coupled to a hydrophobic residue.
 23. Thecomposition according to claim 19, wherein the composition is in a formof a cream, a gel, an unguent, a lotion, a spray, an aerosol, asolution, in an emulsion, in liposomes or in microcapsules, whereinpreferably the composition is for skin treatment.
 24. The compositionaccording to claim 19, further comprising a pharmaceutical acceptablecarrier, filler, bulking agent, disintegrant, stabilizer, binder,humectant, extender, emulsifying agent, dissolution retarder, absorptionenhancer, preservative, antioxidant, wetting agent, adsorbent, lubricantor a combination thereof.
 25. Use of the composition according to claim31 for treatment and/or prevention of a disease, diagnosis of diseases,as a research tool, as a targeting system, as a pharmaceuticalcomposition or as a cosmetic composition.
 26. Use of the compositionaccording to claim 31 in the preparation of a medicament for treatment,prevention and/or diagnosis of a disease.
 27. A method for manufacturingthe composition according to claim 31, comprising at least the followingsteps: providing a substance comprising a cell-penetrating compoundcomprising a basic amino functional group, and adjusting the pH of thecomposition to at least 7.7.
 28. A kit comprising in (a) suitablecontainer(s) at least a composition according to claim 31, andoptionally a package insert.
 29. The kit according to claim 28, whereinthe composition is contained in a ready-to-use form.
 30. The methodaccording to claim 1, further comprising a step of applying thesubstance to a subject, wherein the method is a cosmetic or therapeuticmethod.
 31. A composition comprising a substance to be delivered tocells, wherein the substance comprises a cell-penetrating compoundcomprising a basic amino functional group, and wherein the pH of thecomposition is at least 7.7.